Currently used methods for enzymatic determination of fecal bile acids are inaccurate since, a) 3-keto bile acids which are present (5-20 percent) in fecal bile acid extracts are not determined; b) there are losses of 7 and 12-keto and 7,12-diketo bile acids during the saponification stage of the assay, and c) extract purfication prior to colorimetry is unsatisfactory, resulting in high blanks. In the proposed method, the keto acids, present in fecal extracts, are reduced to alpha and beta hydroxy bile acids prior to the saponification stage of the assay. The resulting extracts are then, a) treated with alkali to effect saponification; b) extracted with petroleum ether to remove non-saponifiable material; c) acidified and extracted with ethyl ether to obtain the "acidic" fraction containing bile acids; d) purified by a three-stage thin-layer chromatography procedure to remove colored impurities, and e) assayed using 3-hydroxysteroid dehydrogenase.